PROJECT SUMMARY Zoonotic coronaviruses (CoVs) such as SARS-CoV and MERS-CoV are pandemic threats. MERS-CoV continues to cause new zoonotic and human transmission and illness with ~35% mortality. Currently, there are no FDA-approved therapies to treat any CoV. New zoonotic CoVs likely will emerge from heterogeneous virus pools in animal reservoirs, thus requiring antiviral strategies aimed at completely conserved and vulnerable targets. CoVs rapidly select for resistance to multiple classes of inhibitors, demonstrating the need for approaches to prevent resistance emergence. Both SARS and MERS infections manifest as severe immunopathologic damage, potentially limiting the therapeutic window for direct-acting antivirals (DAAs). Immunomodulation in the absence of antivirals has been shown to not be beneficial and to even exacerbate SARS and MERS disease. Thus, combinations of DAAs and targeted immunomodulators may be necessary for effective treatment of established infection. The overall goal of our program is to develop CoV antiviral strategies that broadly inhibit known and future potential pandemic zoonotic CoVs, prevent emergence of resistance, and extend the therapeutic window by targeting host immunopathologic responses. The proposed research will advance preclinical development of the CoV-inhibitory nucleoside analogue EIDD- 1931/2801 and other nucleoside analogues in the pipeline and test two small-molecule hits identified as highly active against SARS-CoV for treatment and prevention of epidemic and pre-emergent CoVs. In Specific Aim 1, the spectrum of antiviral activity and therapeutic efficacy of compounds will be defined. The antiviral efficacy, metabolism, and cytotoxicity of each compound will be determined in cultures of primary human lung cells targeted by SARS- and MERS-CoV. The prophylactic and therapeutic efficacy of lead compounds will be evaluated in young, aged, and immunosuppressed murine models of SARS and MERS pathogenesis. In Specific Aim 2, the mechanism of action of lead compounds and kinetics of drug resistance will be determined. The antiviral effect of compounds on virus replication, fidelity, and induction of innate immunity will be assessed. Resistance mutations in genomes of MERS- and SARS-CoV passaged in the presence of increasing concentrations of drug will be determined by deep sequencing. The impact of resistance on SARS- and MERS- CoV virulence, sensitivity to other drugs, and therapeutic efficacy of lead compounds will be determined. Specific Aim 3 will focus on the development of combination regimens for the treatment of emerging CoVs. The combined therapeutic efficacy of DAAs against infections with both wild-type and drug-resistant SARS- and MERS-CoV will be defined using cultured cells and mice. The therapeutic effect of treatment combining a DAA with an immunomodulator will be assessed in mouse models of SARS and MERS. These studies will generate mechanistic and efficacy data necessary for IND filing and origination of human clinical trials.